Individuals with a chronic hepatitis B virus (HBV) infection of the liver have an increased risk for developing hepatocellular carcinoma (HCC). Many aspects of the HBV lifecycle are undefined, and how HBV infections contribute to HCC development is incompletely understood. Although continual immune-mediated destruction of HBV-infected hepatocytes and concomitant liver regeneration are important factors in the development of HBV-associated HCC, HBV proteins such as the HBV X protein (HBx) are likely to contribute to this process. HBx activates numerous cellular signal transduction pathways and stimulates HBV replication. HBx also elevates cytosolic calcium (Ca2+) levels and activates Ca2+ signaling pathways. We previously proposed that HBx activation of Ca2+ signaling is an essential HBx function that is required for HBV replication and analyzed the consequence of this HBx activity in various experimental systems. We have linked HBx regulation of Ca2+ signaling to HBx stimulation of HBV replication and HBx modulation of transcription, proliferation, and apoptosis pathways. We have also demonstrated that HBx can localize to mitochondria and that HBx modulation of Ca2+ signaling is dependent on activities of the mitochondrial permeability transition pore; however, the precise molecular mechanisms that underlie HBx modulation of Ca2+ signaling remain undefined. Although discrepant HBx activities have been observed in different experimental systems, studies in HBV- and HBx-transgenic mice and cultured primary hepatocytes have begun to clarify HBx activities in biologically relevant systems. In studies outlined in this proposal, we will use HBV- and HBx-transgenic mice and cultured primary hepatocytes as model systems to characterize HBx activities. In Aims 1 and 2, we will use primary rat and human hepatocytes to analyze how HBx affects mitochondrial Ca2+ and oxidative stress responses, the consequence of this for factors that associate with mitochondria, and how this impacts HBV replication and hepatocyte apoptosis. HBx effects in cultured hepatocytes will be compared to effects identified in mRNA and miRNA microarray profile databases of liver biopsies from HBV-associated liver diseases to compare observations in cultured hepatocytes to observations in HBV-associated liver diseases in humans. In Aim 3, we will use HBV- and HBx-transgenic mice to study the impact of HBx and HBV replication on intercellular Ca2+ signaling and oxidative stress in the whole liver. We will also examine how HBx modulation of intercellular Ca2+ signaling and oxidative stress impacts in vivo HBV replication. We hypothesize that HBx modulates mitochondria Ca2+ to regulate cytosolic Ca2+ levels and stimulate HBV replication and that a consequence of this HBx activity is to alter mitochondrial functions, eventually disrupting intracellular and intercellular signaling and contributing to processes involved in HCC development. Our longterm goal is to characterize HBx activities and how these impact HBV replication, hepatocyte physiology, and HCC development. Our studies may also identify novel therapeutic targets that could inhibit HBV replication and associated HCC development.